This project focuses on the distribution of specific tobacco carcinogen-DNA adducts within the two genes frequently targeted in smoking induced lung cancer: the p53 tumor suppressor gene and the K-ras proto-oncogene. Lung tumors of smokers often contain characteristic "hotspots" for point mutations within K-ras codon12 and p53 codons 157,158, 248, and 273. These mutations are believed to be a result of DNA polymerase errors during the replication of DNA chemically modified by metabolically activated tobacco carcinogens. The large numbers of mutations at specific sites of the K-ras and p53 genes may originate from their high reactivity towards tobacco carcinogens, deficient repair of DNA lesions, or elevated mispairing rates for the lesions within specific sequence context. While the mispairing rates of DNA lesions introduced in a defined sequence environment can be accurately established by site specific mutagenesis, analytical methods capable of mapping the formation and repair of specific DNA lesions within gene sequences are lacking. The present study will use a mass spectrometry based approach recently developed in this laboratory to quantify the formation of DNA adducts at specific positions within p53 and K-ras-derived DNA sequences. The research will focus on DNA damage induced by two prominent tobacco carcinogens, benzo[a]pyrene(B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Double-stranded synthetic oligodeoxynucleotides representing mutation-prone regions of the p53 and K-ras genes will be treated with reactive metabolites of B[a]P and NNK, and the extent of adduct formation at each position will be determined by liquid chromatography-electrospray ionization tandem mass spectrometry in combination with stable isotope labeling. The same approach will be used to analyze the effect of endogenous cytosine methylation on the formation of tobacco carcinogen-induced guanine lesions and to determine the effect of K-ras and p53 sequence context on the repair of NNK-induced O6-alkylguanine adducts. These studies will 1) Establish the distribution of B[a]P diolepoxide-induced nucleobasel esions within K-ras and p53-derived DNA sequences containing known mutational hotspots. 2) Map the formation of NNK-induced methylated and pyridyloxobutylated lesions along K-ras and p53-derived DNA sequences. 3) Analyze the effects of endogenous cytosine methylation on the formation of B[a]P and NNK adducts at neighboring guanines. 4) Examine the effects of K-ras sequence context on O6-alkylguanine DNA alkyltransferase-catalyzed repair of NNK-induced O6-alkylguanine lesions. The results of this work will afford new insights into the molecular basis of genetic changes observed in smoking-induced lung cancer and will aid in the development of rational prevention strategies and mechanism-based biomarkers for individuals at risk. This research will also lay a foundation for future in vivo studies of carcinogen-modified nucleobases at single nucleotide resolution.